Cells In Inner Nuclear Layer Research Articles

The Expression of irx7 in the Inner Nuclear Layer of Zebrafish Retina Is Essential for a Proper Retinal Development and Lamination

Irx7, a member in the zebrafish iroquois transcription factor (TF) family, has been shown to control brain patterning. During retinal development, irx7's expression was found to appear exclusively in the inner nuclear layer (INL) as soon as the prospective INL cells withdraw from the cell cycle and during retinal lamination. In Irx7-deficient retinas, the formation of a proper retinal lamination was disrupted and the differentiation of INL cell types, including amacrine, horizontal, bipolar and Muller cells, was compromised. Despite irx7's exclusive expression in the INL, photoreceptors differentiation was also compromised in Irx7-deficient retinas. Compared with other retinal cell types, ganglion cells differentiated relatively well in these retinas, except for their dendritic projections into the inner plexiform layer (IPL). In fact, the neuronal projections of amacrine and bipolar cells into the IPL were also diminished. These indicate that the retinal lamination issue in the Irx7-deficient retinas is likely caused by the attenuation of the neurite outgrowth. Since the expression of known TFs that can specify specific retinal cell type was also altered in Irx7-deficient retinas, thus the irx7 gene network is possibly a novel regulatory circuit for retinal development and lamination.

Valproic Acid Regulates Antioxidant Enzymes and Prevents Ischemia/Reperfusion Injury in the Rat Retina

Purposes: To investigate whether valproic acid (VPA) has a neuroprotective effect against ischemia/reperfusion (I/R) injury in the rat retina, and to elucidate the potential antioxidant mechanisms involved.Methods: Adult male Wistar rats were randomly divided into four groups: sham (group A), sham plus VPA (group B), I/R plus vehicle (group C), and I/R plus VPA (group D). Retinal I/R injury was produced by inducing an exceedingly high intraocular pressure (IOP). Prior to insult, VPA was administered subcutaneously (300 mg/kg twice daily) for 7 days, after which the animal was sacrificed. Levels of retinal malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were determined. Protein expressions of retinal poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were analyzed by Western blotting 24 h after injury. Apoptosis of retinal cells was evaluated 24 h after I/R injury by immunofluorescence of activated caspase-3 in histological sections of retina. Seven days after reperfusion, electroretinography (ERG) was performed, and retinal histological changes were examined by light microscopy.Results: Following ischemia, the thickness of the entire retina, including the inner nuclear layer (INL) and inner plexiform layer (IPL), as well as the number of cells in the ganglion cell layer (GCL) were significantly greater in group D than in group C (p < 0.05). VPA suppressed I/R-induced reductions in ERG a- and b-wave amplitudes (p < 0.05). VPA attenuated I/R-induced activation of caspase-3 in ganglion cells and INL cells (p < 0.001). VPA significantly decreased MDA levels and increased activities of SOD, GSH-Px, and CAT in group D (p < 0.05). VPA attenuated activation of PAR and accumulation of NT in the retina after I/R (p < 0.01).Conclusions: VPA protects the retina from I/R injury by enhancing anti-oxidative effects and inhibiting apoptosis of retinal cells.

TRP channel gene expression in the mouse retina

In order to identify candidate cation channels important for retinal physiology, 28 TRP channel genes were surveyed for expression in the mouse retina. Transcripts for all TRP channels were detected by RT-PCR and sequencing. Northern blotting revealed that mRNAs for 12 TRP channel genes are enriched in the retina. The strongest signals were observed for TRPC1, TRPC3, TRPM1, TRPM3, and TRPML1, and clear signals were obtained for TRPC4, TRPM7, TRPP2, TRPV2, and TRPV4. In situ hybridization and immunofluorescence revealed widespread expression throughout multiple retinal layers for TRPC1, TRPC3, TRPC4, TRPML1, PKD1, and TRPP2. Striking localization of enhanced mRNA expression was observed for TRPC1 in the photoreceptor inner segment layer, for TRPM1 in the inner nuclear layer (INL), for TRPM3 in the INL, and for TRPML1 in the outer plexiform and nuclear layers. Strong immunofluorescence signal in cone outer segments was observed for TRPM7 and TRPP2. TRPC5 immunostaining was largely confined to INL cells immediately adjacent to the inner plexiform layer. TRPV2 antibodies stained photoreceptor axons in the outer plexiform layer. Expression of TRPM1 splice variants was strong in the ciliary body, whereas TRPM3 was strongly expressed in the retinal pigmented epithelium.

Effects of PRD supermicropowder on mitochondrial pathway of retinal neuron apoptosis in diabetic rat

Background Research demonstrated that mitochondrial pathway plays a key role in cell apoptosis.Purendan supermicropowder(PRD),a traditional Chinese medicine,may be a potentially effective therapy for neuron apoptosis in diabetic retina.Objective This study was carried out to investigate the effects of PRD on aldose reductase(AR)activity,neuron apoptosis and mitochondrial pathway in retina of diabetic rat.Methods Thirty-six clean male Wistar rats were randomly divided into normal control group,diabetes model group,PRD treatment group randomly and 12 rats for each group.The diabetes models were established by intraperitoneal injection of 25 mg/(kg · d)streptozotocin(STZ)for 3 consecutive days,and blood glucose ≥ 16.7 mmol/L was taken as the standard.PRD solution of 1.8 g/(kg · d)was lavaged in 12 models for 3 months.The eyeballs were enucleated for the preparation of retinal tissue homogenate and slice.AR activity in the retina was detected by ultraviolet spectrophotometry,and neuron apoptosis in retina was assayed by TUNEL staining.Western blot was used to assess the expressions of bcl-2,bax,cyt-c and caspase-3 protein in the retina.The use of animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee(Version 1988).Results Statistically significant differences were found in AR activity and AI among the normal control group,diabetic group and PRD groups(F=90.115,165.540,P＜0.01),and those of diabetic group were evidently higher than the normal control group and PRD group(P＜0.01,P＜0.01).The positive TUNEL cells mainly located in inner nuclear layer and retinal ganglion cell layer.The expressions of bax,cyt-c,caspase-3,bcl-2 and bcl-2/bax in retina were obviously different among these three groups(F =51.332,41.262,25.888,38.564,47.870,P＜0.01),and the expression of bax,cyt-c and caspase-3 protein in diabetic group evidently elevated in comparison with the normal control group and PRD group(t ＝ 10.32,11.04,6.91,P ＜ 0.01)and the expressions of bcl-2 protein and bcl-2/bax value were significantly lower in diabetic rats than in the normal control rats(t =18.05,12.23,P＜0.01).AR activity by AI of retina,the expressions of bax,cyt-c and caspase-3 proteins in retina were obviously lower in PRD group than in diabetes model rats(P ＜ 0.01),and the expression of bcl-2 protein and bcl-2/bax value were significantly higher in PRD group than in diabetes group(P＜0.01).Conclusions PRD can protect retina against the damage caused by high glucose by suppressing AR activity by downregulating the expressions of bax,cyt-c,caspase-3 proteins,increasing the expressions of bcl-2 protein in retina of diabetic rats and further inhibiting the mitochondrial pathway and reducing cell apoptosis in retina of diabetic rats. Key words: Traditional Chinese medicion/purendan supermicropowder; Retina/diabetic retinopathy; cell apoptosis; Mitochondria

Two types of tyrosine hydroxylase-immunoreactive neurons in the zebrafish retina

The purpose of the present study is to identify the dopaminergic amacrine (DA) cells in the inner nuclear layer (INL) of zebrafish retina through immunocytochemistry and quantitative analysis. Two types of tyrosine hydroxylase-immunoreactive (TH-IR) cells appeared on the basis of dendritic morphology and stratification patterns in the inner plexiform layer (IPL). The first (DA1) was bistratified, with branching planes in both s1 and s5 of the IPL. The second (DA2) was diffuse, with dendritic processes branched throughout the IPL. DA1 and DA2 cells corresponded morphologically to A on −s1/s5 and A diffuse −1 ( Connaughton et al., 2004). The average number of total TH-IR cells was 1088 ± 79 cells per retina ( n = 5), and the mean density was 250 ± 27 cells/mm 2. Their density was highest in the mid central region of ventrotemporal retina and lowest in the periphery of dorsonasal retina. Quantitatively, 45.71% of the TH-IR cells were DA1 cells, while 54.29% were DA2 cells. No TH-IR cells expressed calbindin D28K, calretinin or parvalbumin, markers for the various INL cells present in several animals. Therefore the TH-IR cells in zebrafish are limited to very specific subpopulations of the amacrine cells.

Expression of classic cadherins and δ-protocadherins in the developing ferret retina

BackgroundCadherins are a superfamily of calcium-dependent adhesion molecules that play multiple roles in morphogenesis, including proliferation, migration, differentiation and cell-cell recognition. The subgroups of classic cadherins and δ-protocadherins are involved in processes of neural development, such as neurite outgrowth, pathfinding, target recognition, synaptogenesis as well as synaptic plasticity. We mapped the expression of 7 classic cadherins (CDH4, CDH6, CDH7, CDH8, CDH11, CDH14, CDH20) and 8 δ-protocadherins (PCDH1, PCDH7, PCDH8, PCDH9, PCDH10, PCDH11, PCDH17, PCDH18) at representative stages of retinal development and in the mature retina of the ferret by in situ hybridization.ResultsAll cadherins investigated by us are expressed differentially by restricted populations of retinal cells during specific periods of the ferret retinogenesis. For example, during embryonic development, some cadherins are exclusively expressed in the outer, proliferative zone of the neuroblast layer, whereas other cadherins mark the prospective ganglion cell layer or cells in the prospective inner nuclear layer. These expression patterns anticipate histogenetic changes that become visible in Nissl or nuclear stainings at later stages. In parallel to the ongoing development of retinal circuits, cadherin expression becomes restricted to specific subpopulations of retinal cell types, especially of ganglion cells, which express most of the investigated cadherins until adulthood. A comparison to previous results in chicken and mouse reveals overall conserved expression patterns of some cadherins but also species differences.ConclusionsThe spatiotemporally restricted expression patterns of 7 classic cadherins and 8 δ-protocadherins indicate that cadherins provide a combinatorial adhesive code that specifies developing retinal cell populations and intraretinal as well as retinofugal neural circuits in the developing ferret retina.

TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells

The ON pathway of the visual system, which detects increases in light intensity, is established at the first retinal synapse between photoreceptors and ON-bipolar cells. Photoreceptors hyperpolarize in response to light and reduce the rate of glutamate release, which in turn causes the depolarization of ON-bipolar cells. This ON-bipolar cell response is mediated by the metabotropic glutamate receptor, mGluR6, which controls the activity of a depolarizing current. Despite intensive research over the past two decades, the molecular identity of the channel that generates this depolarizing current has remained elusive. Here, we present evidence indicating that TRPM1 is necessary for the depolarizing light response of ON-bipolar cells, and further that TRPM1 is a component of the channel that generates this light response. Gene expression profiling revealed that TRPM1 is highly enriched in ON-bipolar cells. In situ hybridization experiments confirmed that TRPM1 mRNA is found in cells of the retinal inner nuclear layer, and immunofluorescent confocal microscopy showed that TRPM1 is localized in the dendrites of ON-bipolar cells in both mouse and macaque retina. The electroretinogram (ERG) of TRPM1-deficient (TRPM1(-/-)) mice had a normal a-wave, but no b-wave, indicating a loss of bipolar cell response. Finally, whole-cell patch-clamp recording from ON-bipolar cells in mouse retinal slices demonstrated that genetic deletion of TRPM1 abolished chemically simulated light responses from rod bipolar cells and dramatically altered the responses of cone ON-bipolar cells. Identification of TRPM1 as a mGluR6-coupled cation channel reveals a key step in vision, expands the role of the TRP channel family in sensory perception, and presents insights into the evolution of vertebrate vision.

Interleukin-1β promotes the expression of pSTAT3 in rat retinal Miiller cells

Objective To observe the influence of interleukin-1β （IL-1β） on the expression of phosphorylated signal transducers and activators of transcription 3 （pSTAT 3） in rat retinal Muller cells. Methods For in vitro study cultured Mtiller cells were treated with IL-1β of different concentrations （0, 0.1, 1, 5 and 10 ng/ml） for 24 hours. For in vivo study, 32 Sprague-Dawley（SD）rats were divided into 4 groups randomly （control group, 100,500 and 1000 ng/ml group） with 8 rats in each group. After 24 hours of injection with phosphate buffered solution （PBS）, or 100, 500, 1000 ng/ml IL-D into the vitreous cavities of the above rats, retinas were harvested. The expressions of pSTAT3 in cultured Muller cells or treated retinas were evaluated by indirect immunofluorescence and western blotting. Results After 24 hours of incubation without IL-1β, pSTAT3 has little expression in cultured Muller ceils, but was up-regulated by 1 ng/ml or higher IL-1β in a dosage-dependent manner （F： 46.64, 43.78; P〈0.01）. pSTAT3 was not expressed in adult rat retina, but was up-regulated by vitreous injection of 100 ng/ml or higher IL-1β in a dosage-dependent manner （F= 73.53, 43.70; P〈0.01）. pSTAT3 expressed mainly in inner nuclear layer and ganglion cell layer. Double-labeling showed that there was no co-staining of pSTAT3 and glial fibrillary acidic protein （GFAP） in retina of control group, but there were many co-stained Muller cells in retinas treated with IL-1β. Conclusions Expression of pSTAT3 in Muller cells could be activated by IL-1β which may represent one pathway link to reactive gliosis. Key words: Interleukin-1beta/administration dosage; Janus kinases/pathophysiology; Muller cell

Melatonin modulates visual function and cell viability in the mouse retina via the MT1 melatonin receptor.

A clear demonstration of the role of melatonin and its receptors in specific retinal functions is lacking. The present study investigated the distribution of MT1 receptors within the retina, and the scotopic and photopic electroretinograms (ERG) and retinal morphology in wild-type (WT) and MT1 receptor-deficient mice. MT1 receptor transcripts were localized in photoreceptor cells and in some inner retinal neurons. A diurnal rhythm in the dark-adapted ERG responses was observed in WT mice, with higher a- and b-wave amplitudes at night, but this rhythm was absent in mice lacking MT1 receptors. Injection of melatonin during the day decreased the scotopic response threshold and the amplitude of the a- and b-waves in the WT mice, but not in the MT1(-/-) mice. The effects of MT1 receptor deficiency on retinal morphology was investigated at three different ages (3, 12, and 18 months). No differences between MT1(-/-) and WT mice were observed at 3 months of age, whereas at 12 months MT1(-/-) mice have a significant reduction in the number of photoreceptor nuclei in the outer nuclear layer compared with WT controls. No differences were observed in the number of cells in inner nuclear layer or in ganglion cells at 12 months of age. At 18 months, the loss of photoreceptor nuclei in the outer nuclear layer was further accentuated and the number of ganglion cells was also significantly lower than that of controls. These data demonstrate the functional significance of melatonin and MT1 receptors in the mammalian retina and create the basis for future studies on the therapeutic use of melatonin in retinal degeneration.

Vsx1 3′ untranlated region-mediated translation difference at differentdevelopmental stages of goldfish embryos

Visual system homeobox-1 (vsx1) is important in retinal progenitor proliferation, differentiation, and function maintenance of bipolar cells in vertebrates. Recent study in Xenopus laevis has shown that vsx1 3' untranslated region (3' UTR) can mediate cell-specific translation of vsx1 mRNA in the bipolar cells of the retinal inner nuclear layer (INL). vsx1 is also transcribed at the early developmental stages prior to eye formation and its spatiotemporal expression patterns are conserved in all the examined vertebrates. In order to determine whether the vsx1 3' UTR has a role in regulating the spatiotemporal expression of vsx1 during early embryogenesis, we constructed a vsx1 UTR-controlled green fluorescent protein (GFP) reporter gene system and examined the GFP expression pattern in goldfish, Carassius auratus, at different developmental stages. Our results indicated that both the vsx1 5' UTR and the vsx1 3' UTR remarkably repressed GFP expression at transcription level but did not regulate tissue-specific translation at early developmental stages. GFP protein was ubiquitously expressed in the embryos injected with GFP-sensors containing vsx1 UTRs before 60 h post-fertilization (hpf). From hatching stage (72 hpf) onwards, however, GFP protein was specifically expressed in the bipolar cells of the retinal INL in the vsx1 3'UTR-GFP-sensor embryos, but was still ubiquitously expressed in the embryos injected with GFP-sensor lacking vsx1 3' UTR. These observations showed a significant difference of vsx1 3' UTR-mediated translation between early and late developmental stages and suggested that vsx1 3' UTR might not be involved in regulating the spatiotemporal expression of vsx1 until hatching stage during embryogenesis.

Histopathology and ultrastructure changes of retina in rats exposed to n-hexane

To investigate the injury in the retina of rats exposed to n-hexane. Thirty-two SD male rats were randomly divided into control group and four n-hexane groups. The rats in the four n-hexane groups inhaled 35.2 g/m3 n-hexane statically for 1, 3, 7 and 14 days respectively (6 rats in every group) while 8 rats in the control group inhaled air. Histopathology and ultrastructure changes of the retina of rats were analyzed. Rats in control group had clear layers of retinal structure, stained evenly and with regular cell shape. Retinal degeneration was observed in the rats exposed to n-hexane for 7 d and 14 d, and aggravated by degrees with time exposed to n-hexane. In the rats exposed to n-hexane for 14 d, the outer segments of photoreceptor were arranged in a confusing order, and topically there appeared dissolution; in the inner segments, mitochondria were swollen or disappeared. Pyknotic chromatin and cytoplasmic edema were observed in the outer nuclear layer. There were degeneration of horizontal cells, bipolar cells and amacrine cells in the inner nuclear layer. Cytoplasmic edema and organelle dissolution were observed in ganglionic cells. In the neurofibromas layer, outer and inner plexiform layers, there was neuron cell tuber edema, and the microfilament and vacuole of synapse decreased. The histopathology and ultrastructure of retina are damaged in the rats exposed to n-hexane, thus leading to ocular fundus disease.

Regulation of Neurogenesis by Interkinetic Nuclear Migration through an Apical-Basal Notch Gradient

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.

Localization and Diurnal Expression of Melanopsin, Vertebrate Ancient Opsin, and Pituitary Adenylate Cyclase-Activating Peptide mRNA in a Teleost Retina

Localization and Diurnal Expression of Melanopsin, Vertebrate Ancient Opsin, and Pituitary Adenylate Cyclase-Activating Peptide mRNA in a Teleost Retina

Expression of stromal cell-derived factor 1 is increased in the retina of experimental diabetic rats

To investigate the expression of stromal cell-derived factor 1 (SDF-1) in retina of experimental diabetic rats. Thirty four healthy male Wistar rats of six to eight weeks old were chosen and divided randomly into 3 groups: normal control (CON, n = 8), diabetes mellitus 2 months (DM2M, n = 14) and 3 months (DM3M, n = 12). Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg). At the end point, immunochemistry and digital photomicrography analysis were employed to localize and quantitate the expression of SDF-1 in the retinas. The ratios of integrated optical density (IOD) to area of interest (AOI) were calculated to evaluate the intensities of positive staining. Values of z and t, as well as P value of each, were determined by Wilcoxon-Mann-Whitney test and Student's t-test, respectively. Immunofluorescent double-staining was used to indicate the co-expression of SDF-1 and hypoxia-inducible factor-1 (HIF-1), a pivotal transcription factor activated by hypoxia. The SDF-1 mRNA of the retina from DM2M (n = 4) and CON (n = 2) was detected by real-time RT-PCR. Copies of SDF-1 mRNA were then adjusted by the copies of beta-actin mRNA in each sample. Differences between groups were compared by a Student's t test. Positive immunohistochemical staining for SDF-1 was found in each group, most of which located in the inner part of the retina, especially at the inner nuclear layer (INL) and ganglion cell layer (GCL). The inter-quartile range of IOD/AOI of SDF-1 was between 0.6 x 10(-2) to 1.5 x 10(-2) in diabetic group, and 1.1 x 10(-3) to 2.4 x 10(-3) in control group (z = -3.359, P < 0.001). There was no significant difference between DM2M and DM3M (t = 0.952, P > 0.05). Co-expression of SDF-1 and HIF-1 was found in some cells in the inner part of the retina of diabetic rats. Copies of SDF-1 mRNA in DM2M were 2.3 x 10(-4) +/- 1.2 x 10(-4), and those in CON were 2.9 x 10(-4) +/- 1.0 x 10(-4). The ratios of them to beta-actin were 3.3 x 10(-2) +/- 8.5 x 10(-3) for DM2M and 1.7 x 10(-2) +/- 5.7 x 10(-3) for CON (t = 2.33, P < 0.05). Possibly promoted by HIF-1, expression of SDF-1 is increased in the retinas of diabetic rats, which may play an important role in the mechanism of diabetic retinopathy.

Regeneration of Inner Retinal Neurons after Intravitreal Injection of Ouabain in Zebrafish

We examined the regenerative capacity of the adult zebrafish retina by intravitreal injection of a low ouabain concentration to rapidly damage the ganglion cell layer (GCL) and inner nuclear layer (INL) with minimal photoreceptor cell damage. By 24 h after ouabain injection, maximal numbers of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-positive cells were detected in the INL and GCL, with low numbers of TUNEL-positive cells in the outer nuclear layer. Immunolabeling revealed that approximately 85% of the HuC/D-positive amacrine and ganglion cells were lost by 7 d post-ouabain injection (dpi). This ganglion cell loss was consistent with the small, but statistically significant, decrease in the optic nerve diameter. The regeneration response began within 1 dpi with increased proliferating cell nuclear antigen (PCNA) expression in both the INL and GCL. By 3 dpi, PCNA expression is primarily restricted to the Müller glia. By 5 dpi, most of the PCNA expression was localized to neuronal progenitors expressing the olig2:egfp transgene rather than the Müller glia. By 7 dpi, the neuronal progenitors began committing to the ganglion cell fate based on the coexpression of the atoh7:EGFP transgene and the zn5 antigen. The regeneration of ganglion and amacrine cells continued until 60 dpi, when they reached 75% of their uninjected control number. This demonstrates that inner retinal damage, without extensive photoreceptor damage, is sufficient to induce a regeneration response that is marked by the Müller glial cells reentering the cell cycle to produce neuronal progenitor cells that regenerate INL and ganglion cells in the zebrafish retina.

Modulation of expression of platelet-derived growth factor by nuclear transcription factor-kappaB in rats retina

To determine whether nuclear transcription factor-kappaB (NF-kappaB) could modulate the expression of platelet-derived growth factor (PDGF) in rat retina induced by interleukin-1beta (IL-1beta) and studied the mechanism of inflammation-associated retinal diseases such as intraocular inflammation and proliferative vitreoretinopathy (PVR). One hundred and twenty eight Sprague-Dawley rats were divided into A, B, C and D groups, the right eyes were served as experimental eyes, and the left eyes as normal controls. Animals received 2 intravitreal injections at 1 hour interval, which included BSS and BSS (group A); BSS and IL-1beta (group B); pyrrolidine dithiocarbamate (PDTC) and BSS (group C); PDTC and IL-1beta (group D). PDTC was one of special inhibitors of NF-kappaB activation. The Rat eyes were examined by slit-lamp before intravitreal injection, 4 and 24 hours after the injection. Degree of intraocular inflammation was graded using a scoring system. Rats were sacrificed 4 and 24 hours after the injection, the expressions of NF-kappaB and PDGF were detected by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). Four hours after the injection, eyes in group B, as compared with group D, showed more severe intraocular inflammatory signs such as photophobia, iris hyperemia, miosis, large amount of fibrin exudates and posterior synechia of iris. Histopathologic examination showed numerous inflammatory cells within the retinal parenchyma, vitreous and ciliary body. Positive cells of NF-kappaB and PDGF in group D, which could be observed within inner nuclear layer and ganglion cell layer, were obviously less than that in group B. The expression of PDGF mRNA was less in group D compared with group B (P < 0.05), however the expression of PDGF-A mRNA was significantly higher than PDGF-B in group B and group D (P < 0.05). Twenty four hours after the injection, intraocular inflammation alleviated gradually, the expression of PDGF and NF-kappaB decreased obviously. These results suggest that NF-kappaB modulates the expression of PDGF in rat retina induced by IL-1beta. Upregulation of PDGF may play an important role in the occurrence of intraocular inflammation and the different isoform of PDGF may have different impact on various ocular diseases.

Neurokinin A and neurokinin B in the human retina

Very recently, the authors found levels of neurokinin (NK) A-like immunoreactivities in the human retina which were more than five times higher than those of substance P (SP). The present study aimed to find out how many of these immunoreactivities can be attributed to NKA and NKB and then the exact distribution pattern of both NKA and NKB was evaluated in the human retina and compared with that of SP. For this purpose, NKA-like immunoreactivities were characterized in the human retina by reversed phase HPLC followed by radioimmunoassay using the K12 antibody which recognizes both NKA and NKB. Furthermore, the retinae from both a 22- and 70-year-old donor were processed for double-immunofluorescence NKA/SP and NKB/SP. The results showed that NKA contributes to approximately two thirds and NKB to approximately one third of the immunoreactivities measured with the K12 antibody. NKA was found to be localized in sparse amacrine cells in the proximal inner nuclear layer, in displaced amacrine cells in the ganglion cell layer with processes ramifying in stratum 3 of the inner plexiform layer and also in sparse ganglion cells. By contrast, staining for NKB was only observed in ganglion cells and in the nerve fiber layer. Double-immunofluorescence revealed cellular colocalization of NKA with SP and also of NKB with SP. Thus, the levels of NKA and NKB are more than three and two times higher than those of SP, respectively. Whereas the distribution pattern of NKA is typical for neuropeptides, the localization of NKB exclusively in ganglion cells is atypical and unique.

AII amacrine cells in the distal inner nuclear layer of the mouse retina

We serendipitously found a distal Disabled-1 (Dab1)-immunoreactive cell in retina of the C57BL/6J black mouse. The somata of these cells are located in the outermost part of the inner nuclear layer (INL). Their processes extend toward the outer plexiform layer (OPL), receiving synaptic inputs from horizontal and interplexiform cells. In the current study, we name this cell the "distal Dab1-immunoreactive cell." Double-labeling experiments demonstrate that the distal Dab1-immunoreactive cell is not a horizontal cell. Rather, the distal Dab1 cell appears to be a misplaced AII cell, by being glycine transporter-1-immunoreactive and by resembling the latter cell in an electron microscopic analysis. A distal Dab1 cell had been reported in the FVB/N mouse retina, a model of retinitis pigmentosa (Park et al. [2004] Cell Tissue Res 315:407-412). However, here, we found this distal Dab1-immunoreactive cell in the adult and normal developing mouse retinas. Hence, we show that such cells do not require the loss of photoreceptors as suggested previously (Park et al. [2004] Cell Tissue Res 315:407-412). Instead, two other pieces of data suggest an alternative explanation sources for distal Dab1 cells. First, we find a correlation between the number of these cells in the left and right eyes Second, developmental analysis shows that the distal Dab1-immunoreactive cell is first observed shortly after birth. At the same time, AII cells emerge, extending their neurites into the inner retina. These data suggest that distal Dab1-immunoreactive cells are misplaced AII amacrine cells, resulting from genetically modulated anomalies owing to migration errors.

Strategies for Retinal Regeneration in Vertebrates: from Fish to Human

We are studying various aspects of retinal regeneration in zebrafish. We developed a light-induced model for retinal degeneration that results in massive rod and cone cell death in the dorsal and central retina and limited rod cell death in the ventral retina. Within four weeks, the rods and cones regenerated from a population of inner nuclear layer (INL) stem cells in the dorsal and central retina and outer nuclear layer (ONL) rod precursor cells in the ventral retina. A microarray experiment, which examined gene expression at five different key times during light damage and subsequent regeneration, identified several clusters of genes that may play various roles in photoreceptor regeneration. We will use this microarray set to identify the molecules that signal the proliferation response to the INL and ONL cells. To examine the breadth of retinal neurons that can be regenerated, we characterized the effects of intraocular ouabain injection. At low ouabain concentrations, damage was primarily restricted to the INL and ganglion cell layers, while higher ouabain concentrations caused significant cell death in all the nuclear layers. Both types of ouabain-damaged retinas regenerated a nearly wild-type laminated retina, which involved a proliferative cell population residing throughout the retina that may correspond to the stem cells within both the INL and the circumferential germinal zone. Regeneration of the retina also restored the vision-dependent behavioral response. Data will be presented on the degeneration and regeneration responses of the ouabain-treated retina and discussed in relation to the light-damaged retina. The Muller glia, which are neuronal support cells, are a source of regenerated neurons in a variety of organisms and degeneration models. In the light-damaged zebrafish retina, the Muller glia expressed a variety of neuronal precursor genes. These changes in Muller cell gene expression were restricted to the central and dorsal retina, suggesting they are related to either the level of photoreceptor cell damage or the INL cell proliferation. In the ouabain-damaged retina, the Muller glia expressed a different set of genes, which suggests they respond differently to the light and ouabain damage. We will discuss the complexity of the Muller glial cell population and their relative contributions during the regeneration of the light-damaged and ouabain-treated retinas. Finally, to test potential signaling molecules involved in various steps of regeneration (inducing stem cell proliferation, activating Muller glial cells, and identifying the damaged neuronal cell population), we are generating transgenic zebrafish lines that express reporter transgenes at these various steps. Our efforts at isolating these transgenic lines and using them to characterize potential signaling molecules will be discussed.

Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish

Teleost fish regenerate retinal cells from a population of inner nuclear layer (INL) stem cells. To characterize photoreceptor regeneration in zebrafish ( Danio rerio), adult albino fish were subjected to constant intense light to cause photoreceptor cell death. Retinal morphometry was performed on histological sections of control and light-lesioned albino retinas to compare the extent of light damage in the ventral, central and dorsal retinal regions. In addition, opsin immunohistochemistry and TUNEL were used to compare photoreceptor cell death in these different retinal areas, while PCNA immunolabeling quantified the cell proliferation that precedes the photoreceptor regeneration. Transgenic albino; Tg( α1-tubulin:egfp) zebrafish were also exposed to the intense light in order to examine regeneration-related gene expression changes. The light-lesioned retinas are characterized by extensive rod and cone photoreceptor cell death in the central and dorsal regions. In contrast, many of the rods and cones survive in the ventral retina. The highest levels of INL cell proliferation, which occurs subsequent to photoreceptor death, correspond to the retinal regions that suffer the greatest levels of photoreceptor damage. In the ventral retina, where photoreceptor cell death is minimal, cell proliferation is confined to the ONL. In addition, EGFP expression from the α1-tubulin promoter is increased in Müller glial cells in the light-damaged central and dorsal retina, while transgene expression in the ventral retina is restricted to small, round INL cells. Furthermore, expression of the HuC/D neuronal antigen is detected in a subpopulation of the Müller cells in the light-damaged superior retinal region. These data demonstrate that adult albino zebrafish display retinal regional differences in photoreceptor cell death and in the regeneration-related INL cell proliferation response. The high levels of INL cell proliferation and α1-tubulin:egfp transgene expression in the Müller cells may be graded in response to the degree of photoreceptor cell death. This suggests that the levels of photoreceptor damage may directly influence cell responses in the underlying retinal layers.